Inflammatory mediators and modulators released in organ culture from rabbit skin lesions produced in vivo by sulfur mustard. III. Electrophoretic protein fractions, trypsin-inhibitory capacity, α₁-proteinase inhibitor, and α₁- and α₂- macroglobulin proteinase inhibitors of culture fluids and serum

This is the third report in a series on the inflammatory mediators and modulators released in organ culture from skin lesions of various ages, which were produced in vivo in rabbits by the military vesicant, sulfur mustard (SM). It describes the electrophoretic protein fractions and trypsin-inhibitory capacities of the various culture fluids and the amounts of α₁-proteinase inhibitor and α-macroglobulin proteinase inhibitors in these fluids. With one-dimensional electrophoresis, the albumin and β-globulin fractions of protein in culture fluids varied little with the development and healing of the SM lesions. These fractions proportionally resembled the corresponding fractions found in serum. The α₁-globulin fraction was proportionally smaller than the corresponding fractions of serum as the lesions healed. The α₂-globulin fraction was proportionally smaller than the corresponding fraction of serum at all stages of lesion development and healing. The γ-globulin fraction was proportionally larger as the lesion healed. With two-dimensional electrophoresis, about 68%, 46%, and 35% of the protein spots in culture fluids from representative 1-day and 6-day SM lesions and normal skin, respectively, matched those from serum. In each case, the large, diffuse, serum ablumin spot represented about two-thirds of the protein present. Thus, gravimetrically, in normal skin and in both developing and healing lesions, the extracellular proteins were 80-90% of serum origin. The trypsin-inhibitory capacity (TIC) per milligram protein in the culture fluids of healing lesions was markedly less than the TIC per milligram protein in the fluids of peak lesions. This decrease correlates well with the decrease found in the α₁-globulin fraction, which contains α₁-antiproteinase (α₁-PI) (and α₁-macroglobulin [α₁M] in rabbits). The α₁PI and the α₁M-α₂M proteinase inhibitors were identified in the culture fluids by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blots, specific antibodies, and the immunoperoxidase technique. The levels of both free and proteinase-complexed α₁PI and αM inhibitors in the culture fluids decreased as the lesions healed. In both developing and healing lesions, at least half of the α₁PI and αM inhibitors seemed to be complexed with proteinases. Thus, serum seems to be a major source of unbounded extracellular protein within acute inflammatory lesions, and serum proteinase inhibitors seem to be the host's major defense against local damage by proteinases from serum, infiltrating leukocytes, and activated fibroblasts.